Circular site-directed mutagenesis

ABSTRACT

The invention provides improved methods of introducing site-directed mutations into circular DNA molecules of interest by means of mutagenic primer pairs. The mutagenic primer pairs are also selected so as to be either completely complementary or partially complementary to each other, wherein the mutation site (or sites) is located within the region of complementarity. A mutagenic primer pair is annealed to opposite strands of a circular DNA molecule containing the DNA sequence to be mutagenized. After annealing, first and second mutagenized DNA strands, each incorporating a member of the mutagenic oligonucleotide primer pair is synthesized by a linear cyclic amplification reaction. After the linear cyclic amplification mediated synthesis step is completed, the reaction mixture is treated with a selection enzyme that digests the parental template strands. After the digesting step, a double-stranded circular DNA intermediate is formed. The double-stranded circular DNA intermediates is transformed in suitable competent host cells and closed circular double-stranded DNA corresponding to the parental template molecules, but containing the desired mutation or mutations of interest, may be conveniently recovered from the transformed cells. The invention also provide kits for site-directed mutagenesis in accordance with methods of the present invention.

This is a continuation of application Ser. No. 08/567,881, filed Dec. 8,1995.

FIELD OF INVENTION

The invention is in the field of molecular biology, more particularly,in the field of the site-specific mutagenesis.

BACKGROUND

Site-directed mutagenesis has proved to be a remarkably useful tool inmolecular biology. Polynucleotides having pre-determined sequences maynow be designed at will. Polymerase chain reaction (PCR) and variousother cyclic amplification reactions have been adapted for use insite-directed mutagenesis. Although site-directed mutagenesis throughPCR (the polymerase chain reaction) is widely used, PCR basedsite-directed mutagenesis techniques, have several shortcomings.

Several problems exist when trying to perform site-directed mutagenesison double-stranded DNA molecules. These problems include strandseparation and selection against the parental (non-mutated) DNA.Efficient strand separation is important because in a typicalsite-directed procedure, a single polynucleotide primer containing thedesired sequence alteration must compete with the much longercomplementary strand for a hybridization site. Both physical andchemical methods for strand separation have been used. Physical methodsinclude the attachment of the DNA strands to a solid phase, such as aplastic bead (Hall, et al. Protein Eng. 4:601 (1991); Hultman, et al.Nucleic Acids Research 18:5107-5112 (1990); Weiner, et al. Gene126:35-41 (1993), or the use of heat as a denaturant (Landt, et al. Gene96:125-128 (1990); Sugimoto Analytical Biochemistry 179.:309-311 (1989).Chemical methods for strand separation usually rely on increasing the pHof the solution containing the DNA duplex (Weiner, et al. Gene 126:35-41(1993).

Following strand separation, the primer is annealed to the parentalstrand and used to initiate DNA replication. After replication a meansmust be used to reduce the parental plasmid DNA contribution of theheteroduplex before or after cell transformation. Both in vivo and invitro methods have been developed for this reduction. Innon-amplification based in vivo site-directed methods, the incorporationof dUTP into parental DNA during growth of the vector can be selectedagainst in dut⁺, ung⁺ E coli cells (Kunkel Proc. Natl. Acad. Sci.(U.S.A.) 82:488-492 (1985). In vitro methods for selection of themutated strand include; i) unique restriction site elimination (Deng, etal. Analytical Biochemistry 200:81-88 (1992), ii), solid phasetechniques (where the parental DNA remains attached to the solid phase;Hultman, et al. Nucleic Acids Research 18:5107-5112 (1990); Weiner, etal. Gene 126:35-41 (1993), and iii) incorporation of modified bases inthe newly replicated DNA (Taylor et al. Nucleic Acids Research13:8765-8785 (1985); Vandeyar, et al. Gene 65:129-133 (1988).

When PCR has been used for site-specific mutagenesis, a strandseparation is accomplished during the high temperature denaturation stepin the cycling reaction. Selection against the parental DNA is usuallyaccomplished by decreasing the amount of starting template andincreasing the number of rounds of cycling. This increase in the numberof cycles has the adverse effect of increasing the rate of spontaneoussecond-site mutations, especially if an error-prone polymerase such asTaq DNA polymerase is used. In a typical experiment, the mutatedfragment is often subcloned from one vector to another. Often, differentantibiotic resistance markers are alternated or the mutated fragment isgel isolated. Descriptions of the use of the polymerase chain reaction(PCR) in site specific mutagenesis can be found in Hall, et al. ProteinEng. 4:601 (1991); Hemsley, et al. Nucleic Acids Research 17:6545-6551(1989); Ho, et al. Gene 77:51-59 (1989); Hultman, et al. Nucleic AcidsResearch 18:5107-5112 (1990); Jones, et al. Nature 344:793-794 (1990);Jones, et al. Biotechniques 12:528-533 (1992); Landt, et al. Gene96:125-128 (1990); Nassal, et al. Nucleic Acids Research 18:3077-3078(1990); Nelson, et al. Analytical Biochemistry 180:147-151 (1989);Vallette, et al. Nucleic Acids Research 17:723-733 (1989); Watkins, etal. Biotechniques 15:700-704 (1993); Weiner, et al. Gene 126:35-41(1993). Yao, et al. PCR Methods and Applications 1:205-207 (1992). Theuse of site-directed mutagenesis is also described in Weiner et al, Gene151:1/9-123(1994).

Given the many different methods of site-directed mutagenesis that arein use, it is clear that no single technique currently available solvesall of the problems associated with the site-directed mutagenesis. Giventhe state of the art, it is clearly of interest to provide researchers(both industrial and academic) with useful new methods of site-directedmutagenesis. To this end, the inventors have developed new techniquesfor site-direct mutagenesis that have an advantageous combination offeatures as compared to other techniques for site-directed mutagenesis.These useful features include: (1) low secondary mutation frequency, (2)high mutation efficiency, and (3) a minimal number of steps, therebypermitting the generation of host cells containing the mutant sequencesin less than 24 hours.

SUMMARY OF INVENTION

The subject invention provides improved methods of site-directedmutagenesis involving linear cyclic amplification reactions. Theinvention provides extremely simple and effective methods of efficientlyintroducing specific mutations of interest into a target DNA.

The invention provides methods of introducing site-directed mutationsinto circular DNA of interest by means of mutagenic primer pairs thatare selected so as to contain at least one mutation site with respect tothe target DNA sequence. The mutagenic primer pairs are also selected soas to be either completely complementary or partially complementary toeach other, wherein the mutation site (or sites) is located within theregion of complementarity of both mutagenic primers.

In the methods of the invention, a mutagenic primer pair is annealed toopposite strands of a circular DNA molecule containing the DNA sequenceto be mutagenized. After annealing, first and second mutagenized DNAstrands, each incorporating a member of the mutagenic primer pair, aresynthesized by a linear cyclic amplification reaction. The first andsecond mutagenized DNA strands synthesized are of sufficient lengths forforming a double-stranded mutagenized circular DNA intermediate. Thelinear cyclic amplification reaction may be repeated for several cyclesso as to generate a sufficient amount of first and second mutagenizedDNA strands for subsequent manipulations. After the linear cyclicamplification mediated synthesis step is completed, the reaction mixtureis treated with a selection enzyme that digests the parental templatestrands, thereby enriching the reaction mixture with respect to theconcentration of first and second mutagenized DNA strands. The digestionstep serves to digest parental strands that have annealed to the newlysynthesized mutagenized DNA strands and parental strands that haveannealed to one another. After the digestion step, the first and secondmutagenized DNA strands are permitted to hybridize to one another so asto form double-stranded circular DNA intermediates. The double-strandedcircular DNA intermediates are transformed into suitable competent hostcells and closed circular double-stranded DNA containing the is desiredmutation or mutations of interest may be conveniently recovered from thetransformed cells.

The template digesting step in the methods of the invention may becarried out in any of a variety of methods involving a selection enzyme.The selection enzyme, e.g., a restriction endonuclease, is an enzymethat digests parental polynucleotides and does not digest newlysynthesized mutagenized polynucleotides, Either template polynucleotidesprior to replication are modified or polynucleotides synthesized duringreplication are modified so that the selection enzyme preferentiallycatalyzes the digestion of the parent template polynucleotide. In oneembodiment of the invention the polynucleotide for mutagenesis is dammethylated double-stranded DNA and the restriction enzyme used to digestparental polynucleotide strands is Dpn I.

Another aspect of the invention is to provide kits for site-directedmutagenesis with high efficiency. The subject kits contain reagentsrequired for carrying the subject methods.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. This figure provides a schematic diagram of an embodiment of thesubject methods of site-directed mutagenesis. Step (A) shows a circularclosed double-stranded plasmid. The "bull's eye" symbol is used toindicate the target for mutagenesis. Step (B) shows the first and secondmutagenic primer annealed to the circular closed double-strandedplasmid. The crosses indicate the mutagenic sites in the mutagenicprimers. The arrows indicate the direction of synthesis. Step (C) showsthe result of DNA synthesis from a linear cyclic amplification step. Thelighter shaded circular regions represent newly synthesized DNA that isadjoined to the mutagenic primers. The arrows indicate the direction ofsynthesis. Step (D) shows the mutagenized DNA strands that remain aftertreatment with a selection enzyme. The first and second mutagenizedstrands are shown as being annealed to form a double-strandedmutagenized circular DNA intermediate. Note the nicks on each strand.Step (E) shows the resultant mutagenized circular double-stranded DNAmolecules that are recovered after transforming competent cells with thedouble-stranded mutagenized circular DNA intermediate. Note that thecrosses in the diagram reflect the mutagenized sites that correspond tothe "bulls eyes" in Step (A).

DEFINITIONS

The term "linear cyclic amplification reaction," as used herein, refersto a variety of enzyme mediated polynucleotide synthesis reactions thatemploy pairs of polynucleotide primers to linearly amplify a givenpolynucleotide and proceeds through one or more cycles, each cycleresulting in polynucleotide replication. Linear cyclic amplificationreactions used in the methods of the invention differ significantly fromthe polymerase chain reaction (PCR). The polymerase chain reactionproduces an amplification product that grows exponentially in amountwith respect to the number of cycles. Linear cyclic amplificationreactions differ from PCR because the amount of amplification productproduced in a linear cyclic amplification reaction is linear withrespect to the number of cycles performed. This difference in reactionprodcue accumulation rates is a result of using mutagenic primers thatare complementary or partially complementary to each other. A linearcyclic amplification reaction cycle typically comprises the steps ofdenaturing the double-stranded template, annealing primers to thedenatured template, and synthesizing polynucleotides from the primers.The cycle may be repeated several times so as to produce the desiredamount of newly synthesized polynucleotide product. Although linearcyclic amplification reactions differ significantly from PCR, guidancein performing the various steps of linear cyclic amplification reactionscan be obtained from reviewing literature describing PCR including, PCR:A Practical Approach, M. J. McPherson, et al., IRL Press (1991), PCRProtocols: A Guide to Methods and Applications, by Innis, et al.,Academic Press (1990), and PCR Technology: Principals and Applicationsof DNA Amplification, H. A. Erlich, Stockton Press (1989). PCR is alsodescribed in many U.S. Patents, including U.S. Pat. Nos., 4,683,195,4,683,202; 4,800,159; 4,965,188; 4,889,818; 5,075,216; 5,079,352;5,104,792, 5,023,171; 5,091,310; and 5,066,584, which are herebyincorporated by reference. Many variations of amplification techniquesare known to the person of skill in the art of molecular biology. Thesevariations include rapid amplification of DNA ends (RACE-PCR),amplification refectory mutation system (ARMS), PLCR (a combination ofpolymerase chain reaction and ligase chain reaction), ligase chainreaction (LCR), self-sustained sequence replication (SSR), Q-betaamplification, and stand displacement amplification (SDA), and the like.A person of ordinary skill in the art may use these methods to modifythe linear cyclic amplification reactions used in the methods of theinvention.

The term "mutagenic primer" refers to an oligonucleotide primer used ina linear cyclic amplification reaction, wherein the primer does notprecisely match the target hybridization sequence. The mismatchednucleotides in the mutagenic primer are referred to as mutation siteswith respect to the mutagenic primer. Thus, during the amplificationreaction, the mismatched nucleotides of the primer are incorporated intothe amplification product thereby resulting in the synthesis of amutagenized DNA strand comprising the mutagenic primer that was used toprime synthesis mutagenizing the target sequence. The term"oligonucleotide" as used herein with respect to mutagenic primers isused broadly. Oligonucleotides include not only DNA but various analogsthereof. Such analogs may be base analogs and/or backbone analogs, e.g.,phosphorothioates, phosphonates, and the like. Techniques for thesynthesis of oligonucleotides, e.g., through phosphoramidite chemistry,are well known to the person ordinary skilled in the art and aredescribed, among other places, in Oligonucleotides and Analogues: APractical Approach, ed. Eckstein, IRL Press, Oxford (1992). Preferably,the oligonucleotide used in the methods of the invention are DNAmolecules.

The term "digestion" as used herein in reference to the enzymaticactivity of a selection enzyme is used broadly to refer both to (i)enzymes that catalyze the conversion of a polynucleotide intopolynucleotide precursor molecules and to (ii) enzymes capable ofcatalyzing the hydrolysis of at least one bond on polynucleotides so asto interfere adversely with the ability of a polynucleotide to replicate(autonomously or otherwise) or to interfere adversely with the abilityof a polynucleotide to be transformed into a host cell. Restrictionendonucleases are an example of an enzyme that can "digest" apolynucleotide. Typically, a restriction endonuclease that functions asa selection enzyme in a given situation will introduce multiplecleavages into the phosphodiester backbone of the template strands thatare digested. Other enzymes that can "digest" polynucleotides include,but are not limited to, exonucleases and glycosylases.

The term "selection enzyme" refers to an enzyme capable of catalyzingthe digestion of a polynucleotide template for mutagenesis, but notsignificantly digesting newly synthesized mutagenized polynucleotidestrands. Selection enzymes may differentiate between template and newlysynthesized polynucleotides on the basis of modifications to either theparental template polynucleotide or modifications to newly synthesizedmutagenized polynucleotides. Selection enzymes suitable for use in thesubject invention have the property of selectively digesting theparental strands of heteroduplexes formed between parental strands andthe first or second mutagenized DNA strands produced in the linearcyclic amplification reaction step. Examples of selection enzymesinclude restriction endonucleases and endoglycosylases.

The term "double-stranded mutagenized circular DNA intermediate" as usedherein refers to double-stranded circular DNA structures formed byannealing the first mutagenized DNA strand formed in the subject methodsto the second mutagenized DNA strand. When a double-stranded mutagenizedcircular DNA intermediate is transformed into a host cell, host cellenzymes are able to repair nicks (and possible small gaps) in themolecule so as to provide a closed circular double-stranded DNA thatcorresponds to the original DNA molecule for mutagenesis that has beenmodified to contain the specific site-directed mutation or mutations ofinterest.

DETAILED DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The invention provides for, among other things, improved methods forsite-directed mutagenesis. The improved methods of site-directedmutagenesis described herein provide for increased efficiency ofmutagenesis and the reduced introduction of secondary mutations. Themethods of the invention involve the use of pairs of complementary (orpartially complementary) mutagenic primers in linear cyclicamplification reactions. The methods of invention require a minimalnumber of DNA manipulations thereby decreasing the time and cost ofobtaining the desired mutants. In many instances, transformantscontaining DNA constructs with desired mutations may be obtained in asingle day (excluding the time to prepare the mutagenic primers).

The methods of the invention may be used to introduce one or moremutations in DNA sequences of interest. The DNA sequences of interestfor modification by the subject mutagenesis methods are necessarily partof a circular DNA molecule, i.e., the template molecule. The methods ofthe invention comprise the steps of annealing a first and secondmutagenic primer to the double-stranded circular molecule formutagenesis. The mutagenic primers are not generally phosphorylated, butmay be 5' phosphorylated. As the DNA molecule for mutagenesis isdouble-stranded, the annealing step is necessarily preceded by adenaturation step. The annealing step is typically part of a cycle of alinear cyclic amplification reaction. After annealing of the mutagenicprimers, first and second mutagenized DNA strands are synthesized fromthe first and second mutagenic primers, respectively. Synthesis of thefirst and second mutagenized DNA strands takes place during thesynthesis phase of a linear cyclic amplification reaction. The firstmutagenized DNA strand produced from the synthesis necessarily comprisesthe first mutagenic primer at its 5' end. Similarly, the secondmutagenized DNA strand comprises the second mutagenic primer. The linearcyclic amplification reaction may be repeated through several cyclesuntil a sufficiency variety of first and second mutagenized DNA strandsare produced for the subsequent manipulations. After the linear cyclicamplification reaction steps, i.e., first and second mutagenized DNAstrand synthesis are completed, the parental template DNA is digested byadding a selection enzyme. The selection enzyme serves to digestparental strand DNA. The parental strand DNA digested may be in the formof heteroduplexes formed between parental strands and the first orsecond mutagenized DNA strands produced in the linear cyclicamplification reaction step. Additionally, the parental strands digestedby the selection enzyme may consist of duplexes formed between parentalstrands. After the digestion step is completed, the first and secondmutagenized DNA strands are annealed to one another so as to produce adouble-stranded mutagenized circular DNA intermediate. Thedouble-stranded mutagenized circular DNA intermediates are subsequentlyused to transform a competent host cell. Transformed host cells may thenbe isolated as colonies and plasmids, i.e., closed circular DNAs,corresponding to the initial DNA molecules for mutagenesis, butcontaining the desired site-directed mutation or mutations, may beisolated from the transformed cells.

The previous paragraph has been primarily concerned with use ofdouble-stranded circular DNAs as targets for mutagenesis. A person ofordinary skill in the art may readily modify the procedure so as toprovide for site directed mutagenesis of circular single-stranded DNAs.In the case of a single-stranded circular DNA molecule for mutagenesis,only the first mutagenic primer is annealed in the initial step. Afterthe first primer is annealed synthesis of the first mutagenized strandproceeds so as to produce a double stranded circular DNA moleculecomprsing a first mutagenized DNA strand and the parentalsingle-stranded template. After the formation of the circular doublestranded molecule, the method may proceed as described in the previousparagraph.

The methods of the invention employ pairs of mutagenic primersconsisting of a first mutagenic primer and a second mutagenic primer.The mutagenic primers are about 20 to 50 bases in length, morepreferably about 25 to 45 bases in length. However, in certainembodiments of the invention, it may be necessary to use mutagenicprimers that are less than 20 bases or greater than 50 bases in lengthso as to obtain the mutagenesis result desired. The first and secondmutagenic primers may be of the same or different lengths; however, in apreferred embodiment of the invention the first and second mutagenicprimers are the same length.

The first and second mutagenic primers contain one or more mutagenicsites, i.e., mismatch locations with respect to the target DNA sequenceto be mutagenized. The mutagenic site (or sites) may be used tointroduce a variety of mutation types into the DNA sequence formutagenesis. Such mutations include substitutions, insertions, anddeletions. The principle of site-directed mutagenesis with singleoligonucleotide primers is well known to the person of ordinary skill inthe art, and can be found, for example, in Sambrook et al., MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring, Cold SpringHarbor, N.Y. (1989) and Wu et al., Recombinant DNA Methodology, AdademicPress, San Diego, Calif. (1989). This information may be used to designthe mutagenic sites in the first and second mutagenic primers employedin the subject methods.

The first and second mutagenic primers of the invention are eithercompletely complementary to each other or partially complementary toeach other. Preferably, the first and second mutagenic primers areselected so as to be completely complementary to each other. When thefirst and second mutagenic primers are partially complementary to eachother, the region of complementarity should be contiguous. Inembodiments of the invention in which the first and second mutagenicprimer are partially complementary to one another, the region ofcomplementarity must be sufficiently large to permit the mutagenicprimers to anneal to the DNA molecule for mutagenesis; preferably,although not necessarily, the region of complementarity is at least 50%of the length of the primer (50% of the larger primer when the first andsecond primer are of different lengths). The mutagenic sites of thefirst and second mutagenic primers are located in or near the middle ofthe primer. Preferably, the mutagenic sites are flanked by about 10-15bases of correct, i.e., non-mismatched, sequence so as to provide forthe annealing of the primer to the template DNA strands for mutagenesis.In preferred embodiments of subject methods, the GC content of mutagenicprimers is at least 40%, so as to increase the stability of the annealedprimers. Preferably, the first and second mutagenic primers are selectedso as to terminate in one or more G or C bases. The first and secondmutagenic primers for use in the subject invention are optionally 5'phosphorylated. 5' phosphorylation may be achieved by a number ofmethods well known to a person of ordinary skill in the art, e.g., T-4polynucleotide kinase treatment. After phosphorylation, thephosphorylated primers must be purified prior to use in the methods ofthe invention so as to remove contaminants that may interfere with themutagenesis procedure. Preferred purification methods are fastpolynucleotide liquid chromatography (FPLC) or polyacrylamide gelelectrophoresis; however, other purification methods may be used. Thesepurification steps are unnecessary when non-phosphorylated mutagenicprimers are used in the subject methods.

First and second mutagenized DNA strands are synthesized by a linearcyclic amplification reaction. The exact parameter of each portion of acycle of the linear cyclic amplification reaction used may vary inaccordance with factors such as the DNA polymerase used, the GC contentof the primers, DNA concentration, etc. Cycle parameters of concerninclude the time of each portion of the cycle (denaturation, annealingsynthesis) and temperature at which each portion of the cycle takesplace. A person of ordinary skill in the art may obtain guidance inoptimizing the parameters of the cyclic amplication reaction step forindividual experiments can be found in publications describing PCR. Thesynthesis phase of the linear cyclic amplification reactions used in thesubject mutagenesis methods should proceed for a length of timesufficient to produce first and second mutagenized DNA strandsequivalent in length (excluding insertions or deletions in the mutagenicprimers) to the circular DNA molecule for mutagenesis. When Pfu DNApolymerase is used to catalyze the linear cyclic amplification reaction,the synthesis phase of the linear cyclic amplification reactionoptimally occurs with a temperature range of 60°-68° C.; highertemperatures will result in the unwanted effect of mutagenic primerdisplacement.

The linear cyclic amplification reaction, i.e., the synthesis reaction,may be catalyzed by a thermostable or non-thermostable polymeraseenzyme. Polymerases for use in the linear cyclic amplifcation reactionsof the subject methods have the property of not displacing the mutagenicprimers that are annealed to the template, thereby producing amutagenized DNA. strand of essentially the same length as the templatefrom which the newly synthesized strand was derived. Preferably, thepolymerase used is a thermostable polymerase. The polymerase used may beisolated from naturally occurring cells or may be produced byrecombinant DNA technology. The use of Pfu DNA polymerase (Stratagene),a DNA polymerase naturally produced by the thermophilic archaePyrococcus furiosus is particularly preferred for use in the linearcyclic amplification reaction steps of the claimed invention. Pfu DNApolymerase is exceptionally effective in producing first and secondmutagenized DNA strands of the appropriate length for formation of thedesired double-stranded mutagenized circular DNA intermediates. Examplesof other enzymes that may be used in linear cyclic amplificationinclude, but are not limited to, Taq polymerase, phage T7 polymerase,phage T4 polymerase, E. coli DNA polymerase I, Vent™ (New EnglandBiolabs, Beverly Mass.) DNA polymerase, Deep Vent™ DNA polymerase (NewEngland Biolabs, Beverly Mass.), Moloney Murine Leukemia Virus reversetranscriptase, and the like. When the DNA molecule for mutagenesis isrelatively long, it may be desirable to use a mixture of thermostableDNA polymerase, wherein one of the DNA polymerases has 5'-3' exonucleaseactivity and the other DNA polymerase lacks 5'-3' exonuclease activity.A description of how to amplify long regions of DNA using thesepolymerase mixtures can be found, among other places, in U.S. Pat. No.5,436,149, Cheng et al., Proc. Natl. Aca. Sci. USA 91:5695-9 (1994), andBarnes Proc. Natl. Aca. Sci. USA 91:2216-2220 (1994). In order todetermine whether or not a given polymerase (or multiple polymerasecomposition) is suitable for use in catalyzing the sythesis step of thelinear cyclic amplification reaction (under a given set of conditions),a simple assay using primers and circular template may be performed soas to determine if primer displacement occurs. Primer displacement mayreadily be detected by performing the gel electrophoresis anaylsis ofthe assay mixture.

Linear cyclic amplification reactions as employed in the methods of theinvention are preferably carried out with the minimum number ofamplification cycles required to produce the desired quantity of firstand second mutagenized DNA strands. Preferably the number of cycles inthe linear cyclic amplification reaction step is 30 cycles or less, morepreferably 20 or less cylces are performed, and even more preferably thenumber of cylces is between 10 and 20 (inclusive). However, thepreferred embodiment of cycles will vary in accordance with the numberof mutations sought to be introduced into the DNA molecule formutagenesis. Generally, the optimum number of reaction cycles willincrease with the complexity of mutations to be introduced into the DNAmolecule for mutagenesis. The use of a large number of amplificationcycles is troublesome because of the introduction of unwanted secondarymutations in the amplified sequences, i.e., mutations other than theintended site-directed mutagenesis target. Many polymerases used inlinear cyclic amplification reactions, especially Taq DNA polymerase,have relatively high error rates, thus increasing the number ofamplification cycles increases the number of secondary mutationsproduced. Prior to the invention, large numbers of amplification cycleswere required for linear cyclic amplification mutagenesis because of theneed to use a relatively low concentration of amplification target. Inthe past, low concentrations of amplification target were required sothat the amount of non-mutagenized product in a reaction mixture wassignificantly smaller than the amount of desired mutagenized productproduced by linear cyclic amplification reactions, thereby reducing thenumber of transformants containing non-mutagenized polynucleotides. Thesubject methods of site-directed mutagenesis enable the use of acomparatively small number of amplification steps because relativelylarge amounts of template may be used without producing an unacceptablyhigh background of unmutagenized DNA molecules. The digestion stepserves to lower the background of unmutagenized DNA molecules. When alow, e.g., 5-10, number of amplification cycles are used in the linearcyclic amplification mutagenesis reaction, the amount of template DNAmolecule for mutagenesis should be increased so that a sufficient amountof mutagenized product is produced.

The methods of the subject invention comprise a "digesting" or"digestion" step in which the DNA molecules for mutagenesis, i.e., theparental template strands, are digested by a reaction catalyzed by anenzyme. This enzyme is referred to as a "selection enzyme." In order toemploy a parental strand digestion step so as to reduce the parentalbackground in site-directed mutagenesis, a polynucleotide modificationstep must be employed prior to the parental strand digestion step. In apolynucleotide modification step for use in the subject methods ofsite-directed mutagenesis, either (1) one or more of the nucleotides ofthe parental template polynucleotides for mutagenesis are enzymatically(or chemically) modified and the first and second mutagenized DNAstrands synthesized during the replication reaction, e.g., the linearcyclic amplification reaction, are not modified or (2) one or more ofthe nucleotides of the first and second mutagenized DNA strandssynthesized during the linear cyclic amplification reaction areenzymatically (or chemically) modified and the nucleotides of theparental template DNA molecules for mutagenesis are not modified. Theprecise modification reaction step selected for use in a givenembodiment of the invention is selected in conjunction with the specificselection enzyme used in the digestion step so that the selection enzymecan digest the parental strand, i.e., the original templatepolynucleotides, and not significantly digest the newly synthesizedfirst and second mutagenized DNA strands.

The modifying step for use in conjunction with a parental stranddigestion step may comprise the process of exposing a DNA molecule formodification to a modifying agent. The modification step may be carriedout before the linear cyclic amplification reaction step or during thelinear cyclic amplification reaction step. The modifying agent may be amethylase enzyme that catalyzes the methylation of a base within thepolynucleotide of interest. Examples of suitable methylases for use inthe invention include dam methylase, dcm methylase, Alu I methylase, andthe like. The modification reaction may take place in vivo or in vitro.In vivo methylation may be conveniently achieved by propagatingpolynucleotides in cells, either prokaryotic or eukaryotic, thatendogenously produce a suitable methylase enzyme. In a preferredembodiment of the invention, in vivo methylation is used to carry outthe modification step. The polynucleotide modification step may also beaccomplished by synthesizing polynucleotides with nucleotides comprisinga modified base, e.g., 6-methyl-ATP, rather than directly modifying apolynucleotide after the polynucleotide has been completely synthesized.When the modification reaction is a methylation reaction and theselection enzyme is a restriction endonuclease that requires methylatedbases for activity, the methylation step is preferably performed invivo. When the selection enzyme is a restriction endonuclease that doesnot cleave its recognition sequence when the recognition sequence of theenzyme is unmethylated, the modification reaction is preferably amethylation reaction performed in vitro by a polymerase catalyzing theincorporation of methylated nucleotides into a newly synthesizedpolynucleotide strand. When the selection enzyme used in the digestionstep is Dpn I, the modification step is preferably the methylation ofadenine to produce 6-methyl adenine (dam methylase) and the methylationreaction preferably takes place in vivo by propagating the DNA formutagenesis as a plasmid in a suitable prokaryotic host cell.

The digestion step involves the addition of a selection enzyme that iscapable of digesting the parental, i.e., nonmutagenized, strands of theDNA molecule for mutagenesis, but does not significantly digest newlysynthesized polynucleotides produced during a linear cyclicamplification mutagenesis. By performing the digestion step, the numberof transformants containing non-mutagenized polynucleotides issignificantly reduced. The parental strand digestion step involvesadding a selection enzyme to the reaction mixture after the linearcyclic amplification reaction has been completed. Selection enzymes maybe restriction endonucleases or other enzymes that are capable ofcatalyzing the digestion, e.g., cleavage, of parental strands in alinear cyclic amplification reaction, but do not significantly digestthe DNA strands newly synthesized during the linear cyclic amplificationreaction step. Restriction endonucleases for use in the parental stranddigestion step are selected so as to be able to cleave the parentalstrands, but not significantly cleave newly synthesized polynucleotides.The restriction endonuclease selected for use in the digestion step may(1) require a specific modification of the parental strand that is notpresent on the first and second mutagenized DNA strands synthesizedduring the linear cyclic amplification mutagenesis reactions or (2) therestriction endonuclease selected for use in the parental stranddigestion step may be unable to digest polynucleotides that have beenmodified in a specific way and the first and second mutagenized DNAstrands synthesized during linear cyclic amplification reaction havesuch a modification (and the parental template polynucleotides, i.e, theDNA molecules for mutagenesis, lack the modification).

Restriction endonucleases are preferred for use as selection enzymes inthe digestion step. A preferred selection enzyme for use in the parentalstrand digestion step is the restriction endonuclease Dpn I, whichcleaves the polynucleotide sequence GATC only when the adenine ismethylated (6-methyl adenine). Other restriction endonucleases suitablefor use in the parental strand digestion step include Nan II, NmuD I,and NmuE I. However, restriction endonucleases for use as selectionenzymes in the digestion step do not need to be isoschizomers of Dpn I.

In other embodiments of the invention, the selection enzymes used in thedigestion step are not restriction endonucleases. Other enzymes for useas selection enzymes include uracil N-glycosylase. Uracil deglycosylasemay be used as a selection enzyme by modifying a DNA molecule formutagenesis to contain one or more uracil bases rather than thymidine.Uracil incorporation preferably occurs in vivo so that uracildeglycosylase may provide for the digestion of parental strands.Polynucleotides may be modified to as to contain thymidine residues by avariety of methods including DNA synthesis with dUTP as a DNA precursoror the replication of DNA in a dut⁻ ung⁻ strain of E. coli.Polynucleotides comprising uracil bases are sensitive todeglycosylation, i.e., digestion, by uracil N-glycosylase and otherenzymes with similar glycosylase activity. The use of uracilN-glycosylase is described, among other places in Kunkel, PNAS USA,82:488-492 (1985).

After the "digestion" step is completed or concurrent with the"digestion" step, i.e., the addition of the selection enzyme, the firstmutagenized DNA strands and the second mutagenized DNA strands areannealed to one another so as to produce a double-stranded mutagenizedcircular DNA intermediate. The formation of double-stranded mutagenizedcircular DNA intermediate takes place in accordance with conventionalprinciples of nucleic acid hybridization and may be performed under avariety of conditions. Conveniently, the annealing of the first andsecond mutagenized DNA strands so as to form a double-strandedmutagenized circular DNA intermediate may take place simultaneously withthe "digesting" step. The formation of the double-stranded circular DNAintermediates may take place in the same reaction vessel in which the"digesting" and/or the linear cyclic amplification reaction step takeplace. The process of forming double-stranded mutagenized circular DNAintermediates should proceed for a period of time sufficient to producea convenient number of double-stranded mutagenized circular DNAintermediates to provide a convenient number of clones in the subsequenttransformation steps. Generally, incubation for one to two hours at 37°C. will be sufficient in most embodiments of the invention. However,these time and temperature parameters may be readily varied by theperson or ordinary skill in the art so as to take into account factorssuch as DNA concentration, the GC content of the DNA molecules, etc.

After the double-stranded mutagenized circular DNA intermediateformation step is completed, the reaction mixture or a portion thereof,may be used to transform competent single-cell microorganism host cells.It is not necessary to perform a ligation reaction prior totransformation of the host cells. The absence of a ligation steprequirement serves to reduce the time and expense required to carry outthe methods of the invention as compared with conventional methods ofsite directed mutagenesis. The host cells may be prokaryotic oreukaryotic. Preferably the host cells are prokaryotic, more preferably,the host cells for transformation are E. coli cells. Techniques forpreparing and transforming competent single cell microorganisms are wellknown to the person of ordinary skill in the art and can be found, forexample, in Sambrook et al., Molecular Cloning: A Laboratory ManualColdspring Harbor Press, Coldspring Harbor, N.Y. (1989), HarwoodProtocols For Gene Analysis, Methods In Molecular Biology Vol. 31,Humana Press, Totowa, N.J. (1994), and the like. Frozen competent cellsmay be transformed so as to make the methods of the inventionparticularly convenient.

Another aspect of the invention is to provide kits for performingsite-directed mutagenesis methods of the invention. The kits of theinvention provide one or more of the enzymes or other reagents for usein performing the subject methods. Kits may contain reagents inpre-measured amounts so as to ensure both precision and accuracy whenperforming the subject methods. Kits may also contain instructions forperforming the methods of the invention. At a minimum, kits of theinvention comprise: a DNA polymerase (preferably Pfu DNA polymerase), aselection enzyme (preferably Dpn I), control primers, and controltemplates. Kits of the invention may contain the following items:individual nucleotide triphosphates, mixtures of nucleosidetriphosphates (including equimolar mixtures of DATP, dTTP, dCTP anddGTP), methylases (including Dam methylase), control linear cyclicamplification primers, bacterial strains for propagating methylatedplasmids (or phage), frozen competent cells, concentrated reactionbuffers, and the like. Preferred kits comprise a DNA polymerase,concentrated reaction buffer, a selection enzyme, a nucleosidetriphosphate mix of the four primary nucleoside triphosphates inequimolar amounts, frozen competent cells, control primers, and controltemplates. The terms "control template" and "control primer" as usedherein refer to circular double-stranded DNA molecules and mutagenicprimers, respectively that are selected to provide for easily detectablesite-directed mutagenesis by the methods of the invention. For example,a control template may comprise a lac Z gene with a point mutation andthe control primers may be designed to introduce a site-directedmutation that "repairs" the point mutation. As the lac Z phenotype iseasily detected on indicator media, e.g., X-gal, the efficiency of themutagenesis protocol may be easily monitored.

The invention having been described, the following examples are offeredby way of illustrating the invention and not by way of limitation.

EXAMPLES

CONTROL REACTIONS

A procedure for carrying out the site-directed mutagenesis of plasmidpWhitescript™ 5.7-k.b. is given below. This procedure may be readilyadapted for the site-directed mutagenesis of other molecules usingdifferent primers. The plasmid pwhitescript™ 5.7-k.b. encodes a mutantlacZ gene with point mutation that produce a lacZ minus phenotype. Theprimers are designed to "repair" this mutation so as produce a plasmidthat gives rise to a lacZ positive phenotype in E. coli grown onindicator medium. Accordingly, pWhitescript™ 5.7-k.b. may be used as acontrol template in the kits of the invention

Setting Up the Reactions

1. Synthesize two complementary oligonucleotides containing the desiredmutation, flanked by normal nucleotide sequence, i.e., first and secondmutagenic primers. Optionally, the primers are 5' phosphorylated and gelpurified prior to use in the following steps.

2. Prepare the control reaction as indicated below:

5 μl of lox reaction buffer

3 μl (3 ng, 0.001 nM) of pWhitescript™ 5.7-k.b.

control template (1 ng/μl)

1.25 μl (125 ng, 22 nM) of oligonucleotide

control primer #1 34-mer (100 ng/μl)!

1.25 μl (125 ng, 22 nM) of oligonucleotide

control primer #2 34-mer (100 ng/μl)!

1 μl of 10 mM DNTP mix (2.5 mM each NTP)

Double-distilled water (ddH₂ O) to a final volume of 50 μl

Then add:

1 μl of native Pfu DNA polymerase (2.5 U/μl)

3. Prepare the sample reaction(s) as indicated below:

A series of sample reactions using various concentrations of dsDNAtemplate ranging from 2 to 8 ng (e.g., 2, 4, 6 and 8 ng of dsDNAtemplate) should be set up in order to determine the optimum amount.

5 μl of 10× reaction buffer

2-8 ng of dsDNA template

125 ng of oligonucleotide primer #1

125 ng of oligonucleotide primer #2

1 μl of 10 mM dNTP mix (2.5 mM each NTP)

ddH₂ O to a final volume of 50 μl

Then add:

1 μl of native Pfu DNA polymerase (2.5 U/μl)

3. overlay each reaction with 30 μl of mineral oil.

                  TABLE I    ______________________________________    Circular Site-Directed Mutagenesis Cycling Parameters    Segment    Cycles  Temperature Time    ______________________________________    1          1       95° C.                                   30 Seconds    2          10-16   95° C.                                   30 Seconds                       50° C.                                   1 minute                       68° C.                                   2 minutes/kb of                                   plasmid length    ______________________________________

Cycling the Reactions and Digesting the Products

1. Thermal cycle each reaction using the cycling parameters are outlinedin Table I.

2. Repeat segment 2 of the cycling parameters 10-16 times, depending onthe type of mutation desired (i.e., 10 cycles for point mutations, 12cycles for single amino acid changes and 16 cycles for multiple aminoacid deletions or insertions)

3. Following linear amplification, place the reaction on ice for 2minutes to cool the reaction to <37° C.

Note In the following digestion step, it is important to insert thepipet tip below the mineral oil overlay when adding the Dpn Irestriction enzyme to the reaction tubes.

4. Add 1 μl of the Dpn I restriction enzyme (10 U/μl) directly to eachamplification reaction below the mineral oil overlay with a pipet tip.

5. Gently and thoroughly mix each reaction mixture by pipetting thesolution up and down several times. Spin down the reaction mixtures in amicrocentrifuge for 1 minute and immediately incubate each reaction at37° C. for 1-2 hours to digest the parental (i.e., the nonmutated)supercoiled dsDNA.

Transforming into Epicurian Coli XL2-Blue Ultracompetent™ cells(available from Stratagene)

The following protocol has been used successfully for transforming E.coli with pBluescript™-derived plasmids encoding ampicillin orchloramphenicol resistance. Transformation ofkanamycin-resistance-encoding plasmids require a 30- to 45- minuteoutgrowth after 10-fold dilution of the ultracompetent cells with SOCmedium (see Media and Reagent Preparation) between steps 3 and 4 of thetransformation protocol-described in Sambrook et al., Molecular Cloning:A Laboratory Manual, Second Edition, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (1989). Other selections may require a number ofsimilar outgrowth periods.

1. Gently thaw the Epicurian Coli XL2-Blue™ ultracompetent cells on ice.For each control and sample reaction to be transformed, aliquotapproximately 50 μl of the ultracompetent cells to a prechilled Falcon®2059 polypropylene tube.

2. Add 1 μl of the Dpn I-treated DNA from each control and samplereaction to separate aliquots of the ultracompetent cells and swirlgently to mix. Incubate the transformation reactions on ice for 30minutes, swirling periodically throughout the incubation.

As an optional step, verify the transformation efficiency of theEpicurian Coli XL2-Blue ultracompetent cells by adding 1 μl of the pUC18control plasmid (0.1 ng/μl) to a 50 μl aliquot of the ultracompetentcells and incubating as indicated above.

3. Heat pulse the transformation reactions for 45 seconds at 45 secondsat 42° C. and then place the reactions on ice for 2 minutes. This heatpulse has been optimized for the Falcon 2059 polypropylene tubes.

4. Immediately plate the transformation reactions as outlined below:

a. Plate the entire volume of the control transformation reaction andonly 5 μl of the pUC18 control transformation reaction (if performed) onLB-ampicillin-methicillin agar plates (see Media and Reagent sectionbelow) that have been spread with 20 μl of 10% (w/v) X-gal and 20 μl of100 mM IPTG.

Note: Do not mix IPTG and X-gal, since these chemicals will precipitate.X-gal should be prepared in dimethylformamide (DMF) and the IPTG shouldbe prepared in filter-sterilized dH₂ O.

b. Plate the entire volume of each sample transformation reaction onagar plates containing the appropriate antibiotic that is conferred bythe plasmid vector being transformed.

5. Incubate the transformation plates at 37° C. for >16 hours.

The expected colony number should be at least 50 colonies. Greater than80% of the mutagenized control colonies should contain the mutation andappear as blue colonies on agar plates containing IPTG and X-gal.

The mutagenesis efficiency (ME) for the pWhitescript 5.7-kb controltemplate is calculated by the following formula: ##EQU1##

    ______________________________________    MEDIA AND REAGENTS    ______________________________________    TE Buffer        10X Reaction Buffer    10 mM Tris-HCl (pH 7.5)                     100 mM KCl    1 mM EDTA        60 mM (NH.sub.4)2SO.sub.4                     200 mM Tris-HCl (pH 8.0)                     20 mM MgCl.sub.2                     1% Triton ® X-100                     100 μg/ml nuclease-free                     bovine serum albumin                     (BSA)    LB Agar (per Liter)                     LB-Ampicillin-Methicillin Agar    10 g of NaCl     (per Liter)    10 g of tryptone (use for reduced    5 g of yeast extract                     satellite colony    20 g of agar     formation)    Add deionized H.sub.2 0 to a                     1 liter of LB agar    final volume of 1 liter                     Autoclave    Adjust pH to 7.0 with 5 N                     cool to 55° C.    NaOH             Add 20 mg of filter-sterilized    Autoclave        ampicillin    Pour into petri dishes                     Add 80 mg of filter-sterilized    (-25 ml/100-mm plate)                     methicillin                     Pour into petri dishes                     (-25 ml/100-mm plate)    SOB Medium (per Liter)                     SOC Medium (per 100 ml)    20.0 g of tryptone                     SOB medium    5.0 g of yeast extract                     Add 1 ml of a 2 M filter-    0.5 g of NaCl    sterilized glucose    Autoclave        solution or 2 ml of 20%    Add 10 ml of 1 M MgCl.sub.2                     (w/v) glucose prior to    and 10 ml of 1 M use    MgS).sub.4 /liter of SOB medium                     Filter sterilize    prior to use    Filter sterilize    ______________________________________

INCORPORATION BY REFERENCE

All patents, patents applications, and publications cited areincorporated herein by reference.

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. Indeed, variousmodifications of the above-described makes for carrying out theinvention which are obvious to those skilled in the field of molecularbiology or related fields are intended to be within the scope of thefollowing claims.

What is claimed is:
 1. A method of mutagenizing a selected DNA molecule,wherein said DNA molecule is double-stranded and circular, and saidmethod comprises:annealing a first mutagenic primer to a first strand ofsaid DNA molecule and a second mutagenic primer to a second strand ofsaid DNA molecule, wherein said first mutagenic primer comprises aregion that is complementary to the second mutagenic primer and whereinsaid first and second mutagenic primers each contain at least onemutation site with respect to said DNA molecule, wherein the mutationsite is located within the region that is complementary between saidfirst and second mutagenic primers; synthesizing by means of a linearcyclic amplification reaction a first mutagenized DNA strand comprisingsaid first mutagenic primer and a second mutagenized DNA strandcomprising said second mutagenic primer, wherein the first mutagenizedDNA strand and the second mutagenized DNA strand form a double-strandedmutagenized circular DNA intermediate; and digesting the non-mutagenizedstrands of said DNA molecule, wherein said digestion is mediated by aselection enzyme.
 2. The method according to claim 1, wherein theselection enzyme is a restriction endonuclease.
 3. The method accordingto claim 1, wherein the linear cyclic amplification reaction iscatalyzed by Pfu DNA polymerase.
 4. The method according to claim 1,wherein the first and second mutagenic primers are 5' phosphorylated. 5.The method according to claim 1, wherein the linear cyclic amplificationreaction is repeated for less than 20 cycles.
 6. The method according toclaim 1, wherein the first and second mutagenic primers are completelycomplementary to each other.
 7. The method according to claim 1, saidmethod further comprising the steps,annealing said first mutagenized DNAstrand and the second mutagenized DNA strand so as to form adouble-stranded mutagenized circular DNA intermediate, and transforminga host cell with said double-stranded mutagenized circular DNAintermediate.